It is desirable to identify useful methods of purifying proteins that do not destroy, or significantly reduce, the biological activity of the protein. Contaminants must be removed from antibody preparations before they can be used in diagnostic applications, therapeutic applications, applied cell biology, and functional studies. Antibody preparations harvested from hybridoma cell lines, for instance, often contain unwanted components, such as high molecular weight aggregates (HMWA) of the antibody produced by the cell line. This formation of aggregates can adversely affect product safety by causing complement activation or anaphylaxis upon administration. Further, aggregate formation may hinder manufacturing processes by causing decreased product yield, peak broadening, and loss of activity.
The most common protein purification methods are predicated on differences in the size, charge, and solubility between the protein to be purified and contaminants. Protocols based on these parameters include affinity chromatography, ion exchange chromatography, size exclusion chromatography, and hydrophobic interaction chromatography. These chromatographic methods, however, sometimes present technical difficulties in the separation of aggregated or multimeric species of antibodies. Techniques such as ion exchange and hydrophobic interaction chromatography, for instance, may induce the formation of aggregates due to an increased protein concentration or the required changes in buffer concentration and/or pH during elution. Further, in several instances antibodies show differences in isoelectric points that are too small to allow for their separation by ion-exchange chromatography. Tarditi, J. Immunol. Methods 599:13-20 (1992). Size exclusion chromatography is cumbersome and results in the significant dilution of the product, which is a hindrance in large-scale, efficiency-based manufacturing processes. Leakage of ligands from affinity chromatography columns can also occur, which results in undesirable contamination of the eluted product. Steindl, J. Immunol. Methods 235:61-69 (2000). Applicants attempted to remove HMWA from an anti-GDF-8 antibody preparation using anion exchange chromatography, cation exchange chromatography, as well as hydrophobic interaction chromatography. However, all of these methods were unable to substantially remove the HMWA from the anti-GDF-8 antibody preparation.
Hydroxyapatite chromatography is a method of purifying proteins that utilizes an insoluble hydroxylated calcium phosphate [Ca10(PO4)6(OH)2], which forms both the matrix and ligand. Functional groups consist of pairs of positively charged calcium ions (C-sites) and clusters of negatively charged phosphate groups (P-sites). The interactions between hydroxyapatite and proteins are complex and multi-mode. In one method of interaction, however, positively charged amino groups on proteins associate with the negatively charged P-sites and protein carboxyl groups interact by coordination complexation to C-sites. Shepard, J. of Chromatography 891:93-98 (2000).
Crystalline hydroxyapatite was the first type of hydroxyapatite used in chromatography, but it was limited by structural difficulties. Ceramic hydroxyapatite (cHA) chromatography was developed to overcome some of the difficulties associated with crystalline hydroxyapatite, such as limited flow rates. Ceramic hydroxyapatite has high durability, good protein binding capacity, and can be used at higher flow rates and pressures than crystalline hydroxyapatite. Vola et al., BioTechniques 14:650-655 (1993).
Hydroxyapatite has been used in the chromatographic separation of proteins, nucleic acids, as well as antibodies. In hydroxyapatite chromatography, the column is normally equilibrated, and the sample applied, in a low concentration of phosphate buffer and the adsorbed proteins are then eluted in a concentration gradient of phosphate buffer. Giovannini, Biotechnology and Bioengineering 73:522-529 (2000). Sometimes shallow gradients of sodium phosphate are successfully used to elute proteins, while in other instances concentration gradients up to 400 mM sodium phosphate have been used with success. See, e.g., Stanker, J. Immunological Methods 76:157-169 (1985) (10 mM to 30 mM sodium phosphate elution gradient); Shepard, J. Chromatography 891:93-98 (2000) (10 mM to 74 mM sodium phosphate elution gradient); Tarditi, J. Chromatography 599:13-20 (1992) (10 mM to 350 mM sodium phosphate elution gradient). While salts such as NaCl have been incorporated into the binding buffer to purify an antibody using hydroxyapatite chromatography, Giovannini, R. Biotechnology and Bioengineering 73:522-529 (2000), salts such as NaCl and (NH4)2SO4 were not known to affect the elution of proteins in hydroxyapatite chromatography. Karlsson et al., Ion Exchange Chromatography, in Protein Purification, VCH Publishers, Inc. (Janson and Ryden eds., 1989).
In several instances, researchers have been unable to selectively elute antibodies from hydroxyapatite or found that hydroxyapatite chromatography did not result in a sufficiently pure product. Junbauer, J. Chromatography 476:257-268 (1989); Giovannini, Biotechnology and Bioengineering 73:522-529 (2000). Applicants unsuccessfully attempted to separate high molecular weight aggregates from an antibody preparation using ceramic hydroxyapatite chromatography and a sodium phosphate elution based on prior art teachings (FIG. 1). Further, harsh elution conditions, when used in an attempt to break the tight binding of a protein to a matrix, are known to destroy the biological activity of a protein. Thus, there is need for efficient methods of removing impurities, such as high molecular weight aggregates, from antibody preparations, which do not destroy the biological activity of the antibodies.